Enveloped viruses deposit their nucleocapsid into a host cell through the viral envelope protein-mediated fusion between viral and cellular membranes. Thus understanding the mechanism of viral fusion is important for developing novel strategies to prevent infection. However, the molecular details of viral protein-mediated fusion have not been fully elucidated. Two principal entry pathways are used by viruses to establish infection: viral fusion proteins are either activated upon binding to receptors at the cell surface or upon entering acidic endosomes. The avian sarcoma and leukosis virus (ASLV) is an excellent model to study infectious pathways used by disparate viruses, because its entry involves sequential receptor-dependent and low pH-dependent steps that culminate in fusion with acidic endosomes. Single virus imaging is a powerful technique that permits tracking individual virions inside a cell. Through time-resolved imaging of single ASLV fusion, our group has previously visualized the lipid and content transfer steps, demonstrating the progression of fusion through key intermediate stages of hemifusion (lipid transfer), small pore formation and pore enlargement (content release). Our preliminary data show that the fate of nascent fusion pores is determined by the site of fusion: pores formed by ASLV at the cell surface do not fully enlarge, whereas pores formed with the limiting membrane of an endosome dilate efficiently, permitting the release of viral content into the cytosol. This vast difference in the propensity of fusion pores to dilate has led to the hypothesis that viruses have adapted to form relatively small pores at designated sites, relying on cellular endocytic machinery to enlarge these pores and initiate infection. To test this hypothesis, we will: (i) define the ASLV entry pathways and determine the preferred acidic organelles in which fusion occurs;(ii) develop tools to detect the formation of small fusion pores and measure the efficacy of their dilation at the cell surface and in different endocytic compartments;and (iii) determine whether viral proteins can contribute to pore enlargement by testing whether the concerted action of multiple fusion proteins is required to promote viral content release into the cytosol. The proposed studies are expected to provide new insights into the mechanism of ASLV fusion and to elucidate the factors governing pore dilation. Data obtained using the ASLV model would provide a conceptual framework for understanding the entry process of human viruses that enter their host cells via the endocytic pathway. Enveloped viruses initiate infection by depositing their genome into a host cell. This process involves fusion of a membrane surrounding the viral core to a cell membrane. Membrane fusion is a complex multi-step reaction mediated by specialized viral fusion proteins. To elucidate this process, we will visualize fusion of single avian sarcoma and leukosis virions with a host cell from its initiation (a local merger of viral and cellular membranes) to completion (release of the viral genome into the cytosol), using fluorescence microscopy. These studies will help to define the entry mechanisms of human viruses and will suggest new strategies to prevent infection.